An enormous number of bacterial genes either have no annotated function or are only assigned to broad functional categories. However, this list includes many genes that play key roles in bacterial growth and survival. In whole genome screens we have found hundreds of such genes in Mycobacterium tuberculosis, the causative agent of tuberculosis. These include genes that are absolutely required for growth under standard laboratory conditions and those that are needed for optimal growth either in an animal model of infection or under in vitro conditions that are thought to mimic those encountered by the pathogen in human hosts. Moreover, we have found that the M. tuberculosis genome encodes more than 150 intergenic non-coding RNAs that appear to play key regulatory roles. Here we intend to use multiple modalities to discover the functions of M. tuberculosis genes that are critical for bacterial growth and survival. Our program consists of three projects. Projects 1 and 2 will investigate the roles of protein-coding genes that are required for bacterial growth, including those that are needed under standard laboratory conditions (Project 1) and those that permit optimal growth only under conditions similar to those found in infected humans. Project 3 will find non-coding RNA genes that must be optimally expressed to produce normal growth. All projects will utilize the resources provided by three technical cores that will provide metabolomic and biochemical (Core B) and proteomic (Core D) expertise and DNA sequencing and analysis (Core C). With the help of management oversight (Core A) and integration software (Core E) along with the extensive history of collaboration among the investigators, we will determine the function of genes and, with the help of Core E, produce and share high quality data and reagents. RELEVANCE: A huge number of bacterial genome sequences have become available yet a substantial fraction of their identified genes have no known function, even after extensive bioinformatic investigation. Here we intend to experimentally define the roles of genes. We will concentrate on a particularly important class, those that are required for the optimal growth of M. tuberculosis. Project 1: The Roles of Genes Critical for Growth in Vitro Project Leader: Dirk Schnappinger DESCRIPTION (provided by applicant): Throughout history, infectious diseases were a leading cause of human death. In the 20th century, social improvements, antimicrobial chemotherapy and immunization led to a brief period in which infectious diseases were viewed as torments of the past. However, the emergence of new infectious agents and the reemergence of old diseases demonstrated that continued awareness, research and development are necessary to limit the impact of infectious diseases on human health. Tuberculosis (TB) remains the second leading cause of human death from an infectious disease. Drug resistant strains of Mycobacterium tuberculosis (Mtb) threaten the success of TB control programs worldwide and new drugs are needed to effectively treat patients suffering from drug resistant TB and to prevent the spread of drug-resistant TB. Genes Mtb requires for growth in vitro and during infections are among the most attractive targets for the development of new drugs. However, approximately a third of Mtbs in vitro essential genes remain of unknown function, which severely limits their value for drug development. The long-term goal of this application is to overcome this limitation for drug development and to increase our understanding of the biological processes that are fundamental to the growth and survival of Mtb. To achieve this we will utilize conditional gene silencing approaches to construct conditional Mtb knockdown mutants that allow the partial inactivation of in vitro essential genes. We will then (i) perform extensive phenotypic characterization to better understand when and why a gene is required for growth and when and if its inactivation is lethal to the pathogen, and (ii) apply a variety of functional genomics approaches to mechanistically characterize gene functions. RELEVANCE: Tuberculosis (TB) is the world's second leading cause of premature human death from an infectious disease. Work outlined in this proposal will directly increase our understanding of essential Mycobacterium tuberculosis gene functions, contribute to the development of new TB drugs, and ultimately help reducing the impact of this disease on global health.